Connective tissue contraction in wound healing : An experimental study in vivo and in vitro

Abstract: Contraction of connective tissue is an important part of tissue repair that reduces the wound space and helps to decrease the formation of scar tissue, both of which are needed to restore tissue integrity. In the present study, certain cellular mechanisms that are active during connective tissue contraction were assessed and the influence of transforming growth factor-.131 on these mechanisms was evaluated in normal rat and protracted mouse contraction. A new double-embedding technique for sectioning of thin tissue membranes was developed and a multisequence template was constructed that allowed quantification of a number of target mRNAs [:rGF -.131, TGF -.13 type li receptor, alpha-SM actin and .13-actin) from 0.5-2 11g of total RNA. The fibroblast-populated collagen lattice model (FPCL) was used for studies of contraction in vitro, and, in vivo, the perforated mesentery models of rat and mouse were employed. Fourier transformation image analysis was used to assess the orientation of actin in mesenteric wound fibroblasts.Studies in vitro showed that rat, but not mouse, macrophage conditioned medium stimulated contraction of FPCL in serum free medium and that supplementation with serum impaired FPCL contraction. In vivo, rat wound fibroblasts expressed alpha-SM actin during closure of perforations, as shown by in situ hybridization. Using quantitative RT-PCR, it was shown that the expression of alphaSM actin was increased lOO-fold in wounded as compared to unwounded tissue, and that TGF -.131, while stimulating closure of perforations, also increased alpha-SM actin mRNA 4-5 fold inwounded tissue of both rat and mouse. However, the expression of alpha-SM actin was considerably higher in the wounded mesenteriesof rats than in those of mice. TGF -.131 was expressed by normal, unstimulated peritoneal macrophages and, for the first time, shown tobe downregulated in activated, wound macrophages in both rat and mouse. The orientation of actin bundles in wound fibroblasts wasderanged in impaired healing in zinc deficiency but unaffected by TGF -.131 in stimulated contrition.In conclusion, the results have shed further light on various cellular activities during connective tissue contraction. A number of differences were found between rats and mice that help to clarify the mechanism of protracted healing in the mouse. The complex interaction of these mechanisms await to be further elucidated.